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Original Article

Cite this article: Cuesta JA, García Raso JE,Abelló P, Marco-Herrero E, Silva L, Drake P(2019). A new species of pea crab from south-western Europe (Crustacea, Decapoda,Brachyura): species description, geographicdistribution and population structure with anidentification key to European Pinnotheridae.Journal of the Marine Biological Association ofthe United Kingdom 99, 1141–1152. https://doi.org/10.1017/S0025315419000018

Received: 24 May 2018Revised: 29 November 2018Accepted: 3 January 2019First published online: 18 February 2019

Key words:Anomia ephippium; Brachyura; distribution;DNA barcoding; European waters; new species;Pinnotheres bicristatus

Author for correspondence:Jose A. Cuesta, E-mail: [email protected]

© Marine Biological Association of the UnitedKingdom 2019

A new species of pea crab from south-westernEurope (Crustacea, Decapoda, Brachyura):species description, geographic distributionand population structure with an identificationkey to European Pinnotheridae

Jose A. Cuesta1, J. E. García Raso2, Pere Abelló3, Elena Marco-Herrero3,

Luis Silva4 and Pilar Drake1

1Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), Avda. República Saharaui, 2, 11519 Puerto Real, Cádiz,Spain; 2Departamento de Biología Animal, Universidad de Málaga, Campus de Teatinos s/n, 29006 Málaga, Spain;3Institut de Ciències del Mar (ICM-CSIC), Passeig Marítim de la Barceloneta 37- 49, 08003 Barcelona, Spain and4Instituto Español de Oceanografía (IEO), Centro Oceanográfico de Cádiz, Puerto Pesquero, Muelle de Levante,s/n, PO Box 2609, E-11006 Cádiz, Spain

Abstract

After the recent detection, by both morphology and DNA barcodes, of the larval stages of anunknown species of pea crab (Pinnotheres sp.) in European waters, adults of this crab are hereinreported and described as a new species. The current known geographic distribution of the spe-cies comprises theGulf of Cádiz in the easternAtlantic and the adjacentMediterraneanwaters ofthe south of the Iberian Peninsula (Alboran Sea), where this crab is well-established inside theanomiid bivalve Anomia ephippium. In the Gulf of Cádiz, the species displayed a relativelyhigh prevalence: on average, 55.6–77.7%, in A. ephippium samples. The dominant demographiccategories of the new species were soft females (61.8–77.0%) with fewer males (17.7–21.10%).Most of the host bivalves carried only one crab; in bivalves harbouring two crabs, heterosexualpairs were collected more frequently than expected by chance, which suggests that they could bemated pairs. A strong correlation between host size and soft female size was found (r = 0.73, P <0.01) indicating that space availabilitywithin hosts seems to be relevant in determining the size ofthe sedentary phase of the new crab species.

Introduction

The Pinnotheridae de Haan, 1833 is a family of brachyuran decapods comprising some 297–312 species (Ng et al., 2008; Ahyong et al., 2011; Palacios Theil et al., 2016), with several generaand subfamilies that need to be revised (Palacios Theil et al., 2016).

Until very recently, four species of the family Pinnotheridae were known to occur inEuropean waters (Becker & Türkay, 2010; Subida et al., 2011), namely Afropinnotheres monodiManning, 1993, a crab originally from Africa, and the native European Nepinnotheres pin-notheres (Linnaeus, 1758), Pinnotheres pectunculi Hesse, 1872 and P. pisum (Linnaeus,1767). For these four species there are data available on their distribution, larval stages andDNA markers (Pérez-Miguel et al., 2019). This information allowed the detection byMarco-Herrero et al. (2018) of some pinnotherid larvae in plankton samples (ProjectECOBOGUE) from the coast of Doñana National Park (Gulf of Cádiz, SW Spain) that didnot belong, either morphologically or according to DNA analyses, to any of the previouslyknown European pinnotherid species. This was considered as an early detection of a new mar-ine invader in European waters. Since DNA analysis of the larvae indicated that the new spe-cies was closer to species of the genus Pinnotheres than to any other Pinnotheridae genus, itwas provisionally named Pinnotheres sp. (see Marco-Herrero et al., 2018; Pérez-Miguel et al.,2019). Since then, the search was begun for their adult stages, in the context of the AFROBIVproject: this is a project to study the pinnotherid species present in the Iberian Peninsula, pay-ing special attention to the effect of A. monodi on their bivalve hosts. Two approaches wereadopted in this search: (1) the collection of a large number of individuals of different possiblehosts, in the geographic area where larvae appeared; and (2) searching in existing crustaceancollections for samples of pinnotherids collected in Iberian waters, for close morphologicalexamination.

During a recent and exhaustive sampling of intertidal bivalves all along the IberianPeninsula coasts, we did not collect any pinnotherid adults of any unidentifiable species(Pérez-Miguel et al., 2019), and we therefore concluded that adult individuals ofPinnotheres sp. would probably be hosted only by subtidal species. A reexamination of thepea crabs (by morphology and DNA barcodes) deposited in the Biological ReferenceCollections (CBR) at the Institut de Ciències del Mar (ICM-CSIC), resulted in their classifica-tion to three out the four known European species, namely N. pinnotheres, P. pectunculi and P.pisum. However, when reviewing specimens from previous studies by Manjón-Cabeza &

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García Raso (1998) one ovigerous female captured in Barbate(Gulf of Cádiz) was identified as Pinnotheres sp. based on DNAbarcodes, and it showed a clearly different morphology fromthat of other European crab species. Also, a soft female livinginside the common saddle oyster Anomia ephippium Linnaeus,1758 (Anomiidae Rafinesque, 1815) had been collected in thesame area in 1995 (García Raso & Manjón-Cabeza, 2002), andalthough it was initially identified as P. pisum, it was actuallyclose to the above-mentioned ovigerous female of Pinnotheressp. Given these findings, we then focused on obtaining samplesof Anomia ephippium from the same area to search for more spe-cimens and confirm the habitat and distribution of this species.

In this study, we analyse by morphology and at the molecularlevel the specimens from Barbate as well as the pea crabs hostedby bivalves collected in two oceanographic surveys conducted in2017 (ARSA 1117 and MEDITS_ES2017) and another survey in2018 (ARSA 0318), paying special attention to A. ephippium.All of these specimens clearly belong to an undescribed speciesof the genus Pinnotheres Bosc, 1801. This new species isdescribed, figured and compared with congeners, and a key forEuropean Pinnotheridae is provided.

Materials and methods

Material examined

The Pinnotheres bicristatus sp. nov. individuals examined in thisstudy correspond to those hosted by subtidal bivalves collectedby the authors during different fishery research cruises alongthe Atlantic and Mediterranean coasts of Andalusia (South ofIberian Peninsula). Samples of Anomia ephippium were obtainedfrom the ARSA 1117, ARSA 0318 and MEDITS_ES 2017 surveysusing bottom trawl gears; an additional pea crab was collectedinside A. ephippium, and another was collected free-living inbivalve samples from offshore the Barbate coast (ProjectPB92-0415) with a dredge (Manjón-Cabeza & García Raso,1998; García Raso & Manjón Cabeza, 2002). The coordinates ofall sampling sites in which Pinnotheres bicristatus sp. nov. hasbeen collected are shown in Table 1, and a map showing the pos-ition of the sampling stations is displayed in Figure 1. Thedescription of the new species in this paper is only by two ofthe authors, J.E. García Raso and J.A. Cuesta.

Collection and measurement of hosts and crabs

The specimens of Anomia ephippium collected during theMEDITS_ES 2017 survey were opened on board the ship and

inspected for the presence of pea crabs, which were preserved in80% ethanol. The A. ephippium collected during the ARSA1117 and ARSA 0318 (hosting 98.4% of crabs examined) were fro-zen in the ship, and later examined in the laboratory, where theywere measured and inspected for the presence of crabs. For A.ephippium from the ARSA cruises, the maximal shell lengthsalong the anterior-posterior and dorsal-ventral axes of the shellwere measured to the nearest 0.1 mm with a dial caliper (TesaCal IP65). Due to the variability in the shell shape, the shellsize of A. ephippium was estimated as the mean values of bothmeasures.

All crabs found within each individual host were preservedand stored in 80% ethanol for further demographic observations.The carapace width (CW =maximum cephalothorax width) andcarapace length (CL = distance from the rostrum to the posteriormargin of the carapace) of each crab was measured to thenearest 0.01 mm under a stereomicroscope provided with a cali-brated ocular micrometer. The wet weight (WW) was estimatedwith a digital balance (precision 0.1 mg). Crabs were sexedbased on the presence (male) or absence (female) of gonopods.Furthermore, each female crab was classified either as hard orsoft by considering its pleonal morphology, cephalothoraxconsistency and shape, and pleopods morphology: hard femalesshow a flattened and rounded cephalothorax and swimminglegs (similar to those of males), whereas the body of soft femalesis mostly sub-trapezoidal and lacks swimming legs. Lastly, softfemales carrying eggs (embryos) were classified as ovigerousfemales. Gonadal development of soft females was considered.The drawings were made using a camera lucida and the photo-graphs using a Nikon digital camera DXM1200F, both mountedon stereomicroscopes. Other abbreviations used: MXP3, thirdmaxilliped; P1–P5, pereiopods 1–5 (P1 – cheliped, P2–P5 – walk-ing legs); IEO-CD, Decapod Crustacean Collection from CadizOceanographic Centre; CBR, Biological Reference Collections.

Data treatment of the Gulf of Cádiz population

For each of the two ARSA surveys, the host-size related prevalenceof Pinnotheres bicristatus sp. nov. in the host Anomia ephippiumwas analysed by estimating the number of crabs collected for each5 mm size group of the host, as well as by representing the cumu-lative number of A. ephippium in size-increasing order vs thecumulative number of crabs retrieved from them. In addition,for each demographic category of crabs, the relationship betweenhost size and crab size was assessed by regression analysis by fit-ting data to a linear model. Similarly, the relationship between

Table 1. Locations, surveys and dates in which the pea crab Pinnotheres bicristatus sp. nov. has been collected

Sampling site Code Coordinates Depth (m) Survey Date

Doñana N.P. coast A1 36°57.5′N 6°32.5′W 11 ECOBOGUE (MT10) 14 November 2013

Doñana N.P. coast A2 36°51.5′N 6°29.0′W 14 ECOBOGUE (PD10) 14 November 2013

Cádiz Bay offshore A3 36°34.8′N 6°27.5′W 28–30 ARSA 0318 (L24) 25 February 2018

Cádiz Bay offshore A4 36°34.1′N 6°26.6′W 23–27 ARSA 1117 (L102) 30 October 2017



Barbate offshore A7 36°13.2′N 6°14.0′W 52 ARSA 0318 (L1) 19 February 2018

Barbate offshore A8 36°09.7′N 5°53.6′W 15–18 Project PB92–0415 22 July 1994

Barbate offshore A8 36°09.7′N 5°53.6′W 15–18 Project PB92–0415 17 March 1995

Estepona offshore M1 36°20.3′N 5°12.9′W 47 MEDITS_ES 2017 (L007) 25 April 2017

Codes of sampling sites as in Figure 1.

1142 Jose A. Cuesta et al.



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crab carapace width and crab wet weight was assessed by regres-sion analysis by fitting data to power regression models.Differences between the estimated slopes of regression lines orbetween the exponents of power models were tested by usingStudent’s t-tests.

To test whether crabs tend to live solitarily, in pairs or aggre-gated in higher numbers inside the host, the frequency of occur-rence of A. ephippium without crabs and with different numbersof crabs was compared with the expected frequencies under a ran-dom distribution (binomial distribution) and differences tested bya χ2 test. Similarly, the observed proportion of males to femaleswas tested for deviation from a 1:1 sex ratio by using a χ2 test.Besides, the observed sexual composition of crab pairs sharing asingle host individual was compared with the expected frequen-cies of heterosexual and homosexual pairs in a random distribu-tion by using χ2 test.

A 5% significance level (P = 0.05) was considered for all statis-tical tests carried out in this study (Zar, 2010).

Pinnotheres bicristatus sp. nov. identification by DNAbarcodes

Total genomic DNA was extracted from pereiopod muscle tissue,following a modified Chelex 10% protocol by Estoup et al. (1996).Partial sequences of the mitochondrial 16S and Cox1, and nuclearH3 genes were amplified. Cycling conditions of the polymerasechain reaction (PCR), length of the sequences obtained, and pri-mers used for each gene are the same as in Pérez-Miguel et al.(2019). PCR products were sent to Stab-Vida laboratories to bepurified and then bidirectionally sequenced. Sequences were

edited using the software Chromas version 2.0. The obtainedfinal DNA sequences were compared with those from Pinnother-idae available in GenBank database.

An evolutionary distances analysis was carried out in MEGA6(Tamura et al., 2013). A concatenated alignment of the 16S, Cox1and H3 sequences was built using the sequences obtained in thepresent study from specimens of Pinnotheres bicristatus sp. nov.,as well as the sequences of A. monodi, N. pinnotheres, P. pectun-culi, P. pisum, Pinnotheres sp. (larvae), Orthotheres barbatus(Desbonne, 1867) and Zaops ostreus (Say, 1817) downloadedfrom GenBank (http://www.ncbi.nlm.nih.gov). The phylogenyreconstruction analyses were inferred from neighbour-joiningusing the p-distance method. The nodal confidence of theobtained topologies was assessed via 2000 bootstrap replicates.

Results

Geographic distribution and host of Pinnotheres bicristatussp. nov.

The known distribution of P. bicristatus sp. nov. is, so far, theGulf of Cádiz (NE Atlantic) and western Alboran Sea (SWMediterranean) coasts (Figure 1). In this study, we have examinedone ovigerous female collected free-living in bivalve samples inBarbate offshore (Gulf of Cádiz) and 328 individuals hosted byAnomia ephippium: 325 specimens collected along the coast ofthe Gulf of Cádiz and 3 specimens collected in the Alboran Sea(Table 2).

Gulf of Cádiz population

Crab prevalence and size and sex structureIn the Gulf of Cádiz, the mean prevalence by Pinnotheres bicris-tatus sp. nov. in its unique known host, Anomia ephippium, was55.6% (48.15–66.67%) in October 2017 and 77.7% (67.2–88.2%)in February 2018.

Neither megalopae nor indeterminate (small individuals with-out sexual characteristics) individuals of P. bicristatus sp. nov.were found inside the host. The three demographic categoriesobserved were males, hard females and soft females (Table 2,Figure 2). In P. bicristatus sp. nov. population from the Gulf ofCádiz (98.4% of the crabs examined), the dominant categorywas soft females (77.0% in October 2017 and 61.8% in February2018), followed by males (17.7% in October 2017 and 21.1% inFebruary 2018) and hard females (5.3% in October 2017 and17.1% in February 2018). The carapace width (and wet weight)of crabs ranged from 2.0 (4.0 mg) to 10.4 mm (354.9 mg), withsoft females showing the largest average size of 7.2 ± 0.1 mm(155.4 ± 5.7 mg) in October 2017 and 5.9 ± 0.1 mm (112.0 ±7.3 mg) in February 2018, followed by males with a mean sizeof 3.9 ± 0.1 mm (55.8 ± 2.4 mg) in October 2017 and 3.5 ±0.1 mm (25.3 ± 2.7 mg) in February 2018, and hard femaleswith mean size of 3.1 ± 0.1 mm (18.1 ± 1.7 mg) in October 2017and 3.1 ± 0.2 mm (16.0 ± 2.4 mg) in February 2018. Most softfemales collected in both surveys showed well developed reddishgonads (98.7% in October and 87.2% in February) and the smal-lest female with this characteristic had a carapace width of 3.5 mm(February 2018).

The sex-ratio (female/male) was: 4.0 in October 2017, whichdiffered significantly from a 1:1 sex ratio (χ2 = 103.2, P < 0.01),and 3.8 in February 2018, which also differed significantly froma 1:1 sex ratio (χ2 = 13.9, P < 0.01). Irrespective of the samplingdate and demographic category, there was a significant (P <0.01) power relationship between carapace width and wet weight(Figure 2). The exponent of fitted models for males (b = 2.979)was very close to 3 (t-student = 0.1; P > 0.05), whereas those of

Fig. 1. Map showing sampling sites where Pinnotheres bicristatus sp. nov. was col-lected. ECOBOGUE 2013: A1, A2, Doñana National Park coast; ARSA 2017: A4, A5,A6 offshore Bay of Cadiz; ARSA 2018: A3, offshore Bay of Cadiz, A7, offshoreBarbate coast; Project PB92–0415: A8, Barbate coast; MEDITS_ES 2017: M1, offshoreEstepona.

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soft females (b = 2.597) and hard females (b = 2.519) were lowerthan 3 although this difference was statistically significant forsoft females (t = 4.3; P < 0.01) but not for hard females (t = 1.0;P > 0.05).

Interactions between crabsThe number of crabs per host varied between 0 and 2, with 96.7%of Anomia ephippium with crabs hosting a single individual inOctober 2017 and 73.3% in February 2018 (Figure 3A). Thus,crab distribution among hosts did not display a random pattern:there were more bivalves harbouring one crab and fewer bivalveswithout crabs or harbouring two crabs, than those expected bychance alone. The observed departure from a random patternwas statistically significant in October 2017 (χ2 = 54.3, P < 0.01)and February 2018 (χ2 = 10.7, P < 0.01). A total of six A. ephip-pium harbouring heterosexual pairs of crabs were collected inOctober 2017 (3.3% of infested hosts); whereas 12 A. ephippiumwith crab pairs (26.7%) were collected in February 2018: 10 het-erosexual pairs, one 2-males pair and one 2-females pair.Considering the sex ratio observed in each survey (Table 2),there were more heterosexual pairs than expected by chancealone both in October 2017 (χ2 = 9.2; P < 0.01) and in February2018 (χ2 = 11.3; P < 0.01). One male and one soft female wasthe combination observed in 55% of heterosexual pairs, whereasone male and one hard female combination was observed in45% of them.

Host size effectsThe collected Anomia ephippium in October 2017 survey had amean size (±SE) of 40.4 (±0.4) mm and ranged between16.6 mm and 55.3 mm, whereas those of February 2018 had amean size of 35.6 (±0.8) mm ranging from 15.2 to 52.0; similarly,the mean size of specimens hosting pea crabs was 40.5 (±0.5) mmin October 2017 and of 36.0 (±0.9) mm in February 2018.

A higher number of crabs was collected in the most frequentsize classes of A. ephippium in both surveys. This homogeneousprevalence of crabs throughout the host size range was evidencedby an increase of the cumulative number of crabs collected pro-portional to that of the cumulative number of hosts when the lat-ter was estimated by ordering hosts by increasing size (Figure 3A).

For crabs collected in the 2017 and 2018 surveys, there was nosignificant correlation between hard female sizes and those oftheir hosts (r = 0.07; P > 0.05), whereas a significant positive cor-relation between host and crab sizes was observed for both soft

females (r = 0.73; P < 0.01) and, to lesser extent, males (r = 0.49;P < 0.01). Nevertheless, soft females hosted by A. ephippium ofa specific size were larger than males hosted by A. ephippium ofthe same size (Figure 3B).

Pinnotheres bicristatus sp. nov. identification by molecularanalysis

All the sequences obtained for the three genes (mithocondrial 16Sand Cox1, and nuclear H3) fit 99–100% with those of Pinnotheressp. larvae (zoeae and megalopa) deposited in GenBank under theaccession codes MF069149 and MF069150 (16S), MF134396(Cox1) and MF138913 (H3) obtained by Marco-Herrero et al.(2018) and Pérez-Miguel et al. (2019). In the final tree, all sequencesof Pinnotheres bicristatus sp. nov. (adults and larvae) clusteredtogether within a clade, separate from the other EuropeanPinnotheres species (P. pisum and P. pectunculi) (Figure 4).

Species description

SystematicOrder DECAPODA Latreille, 1802

Infraorder BRACHYURA Latreille, 1802Family PINNOTHERIDAE De Haan, 1833

Genus Pinnotheres Bosc, 1801Pinnotheres bicristatus sp. nov. García Raso & Cuesta

(Figures 5–8 & 9G)Pinnotheres sp.: Manjón-Cabeza & García Raso (1998), oviger-

ous female; Pinnotheres pisum: García Raso & Manjón-Cabeza(2002), soft female; Pinnotheres sp.: Marco-Herrero et al.(2018), zoea II, zoea III, zoea IV and megalopa.

Material examinedHolotype: ♂ (4.4 mm CW) (IEO-CD-ICMAN/2419, GenBankaccession numbers MK415384, MK426944 and MK468907) inAnomia ephippium Linnaeus, 1758 (Bivalvia, Anomiidae),36°34.1′N 6°26.6′W, Gulf of Cádiz, 23–27 m depth, detritic bot-tom, coll. Luis Silva (ARSA cruise 1117, 30 October 2017).

Paratypes: in Anomia ephippium Linnaeus, 1758 (Bivalvia,Anomiidae), 36°34.1′N 6°26.6′W, Gulf of Cádiz, 23–27 m depth, det-ritic bottom, coll. Luis Silva (ARSA cruise 1117, 30 October 2017):1 ♀ (hard) (3.27 mm CW) (IEO-CD-ICMAN/2420), 1♂ and 1♀(soft) (3.8 and 7.5 mm CW) (IEO-CD-ICMAN/2421–2422), 1 ♀(soft) (7.35 mm CW) (IEO-CD-ICMAN/2423); 1 ♀ (soft)

Table 2. Characteristics of pea crabs, Pinnotheres bicristatus sp. nov., examined in this study

Host Site Sexual category No. of crabs Mean CW ± SE CW range Mean WW±SE WW range

A. ephippium A4, A5, A6 Fh 13 3.1 ± 0.1 2.3–3.5 18.1 ± 1.7 1.4–261.0

Fr 191 7.2 ± 0.1 4.3–10.4 155.4 ± 5.7 26.1–354.9

M 44 3.9 ± 0.1 2.3–5.0 55.8 ± 2.4 4.0–67.6

A3, A7 Fh 13 3.1 ± 0.2 2.0–4.1 16.0 ± 2.4 6.9–46.0

Fr 47 5.9 ± 0.1 3.9–7.5 112.0 ± 7.3 15.8–223.6

M 16 3.5 ± 0.1 2.8–4.3 25.3 ± 2.7 14.6–38.6

A8 Fr 1 6.5 – 123.6 –

M1 Fr 1 7.1 – 124.8 –

Fo 1 7.2 – 195.9 –

M 1 3.9 – 23.4 –

Not known A8 Fo 1 8.2 226.0 –

CW, crab carapace width (mm); WW, crab wet weight (mg); Fh, hard females; Fr, reproductive females; M, males.Codes and locations of sampling sites (A3 to A8, and M1) as in Figure 1.




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(7.8 mm CW) (IEO-CD-ICMAN/2424), 1 ♀ (soft) (7.50 mm CW)(IEO-CD-ICMAN/2425), 1 ♂ and 1 ♀ (soft) (3.8 and 5.08 mm CW)(IEO-CD-ICMAN/2426–2427), 1 ♀ (7.88 mm CW) (IEO-CD-ICMAN/2428), 1 ♀ (soft) (8.03 mm CW) (IEO-CD-ICMAN/2429),1 ♀ (soft) (6.89 mm CW) (IEO-CD-ICMAN/2430), 1 ♀(soft)(8.26 mm CW) (IEO-CD-ICMAN/2431), 1 ♀ (soft) (5.68 mmCW) (IEO-CD-ICMAN/2432), 1 ♀ (soft) (7.42 mm CW) (IEO-CD-ICMAN/2433), 1 ♂ (3.6 mm CW) (IEO-CD-ICMAN/2434),1 ♀ (soft) (5.76 mm CW) (IEO-CD-ICMAN/2435), 1 ♂ (3.8 mmCW) (IEO-CD-ICMAN/2436). In Anomia ephippium, 36°34.8′N6°27.5′W, Gulf of Cádiz, Spain, 28–30 m depth, detritic bottom,coll. Luis Silva (ARSA expedition 0318, 25 February 2018): 1♀(hard) (2.59 mm CW) (IEO-CD-ICMAN/2437), 1 ♂ and 1 ♀(hard) (3.6 and 2.9 mm CW) (IEO-CD-ICMAN/2438–2439). InBarbate offshore, 36°09.7′N 5°53.6′W, free in bivalve samples of det-ritic bottom, 15–18 m depth, 22 July 1994, coll. Manjón-Cabeza andGarcía Raso (1988, as Pinnotheres sp.): 1 ovigerous ♀ (8.2 mm CW)(IEO-CD-ICMAN/2440, GenBank accession numbers MK426940and MK468903). In Anomia ephippium, Barbate offshore, 36°09.7′N 5°53.6′W, detritic bottom, 15–18 m depth, 17 March 1995,coll. García Raso and Manjón Cabeza (2002, as Pinnotheres pisum):1 ♀ (soft) (6.5 mm CW) (IEO-CD-ICMAN/2441). In Anomiaephippium, Estepona offshore, 36°20.3′N 5°12.9′W, 47 mdepth, coll. Pere Abelló (MEDITS_ES 2017, 25 April 2017): 1 ♂(3.9 mm CW) (ICMD002583, GenBank accession numbersMK415382, MK426942 and MK468905), 1 ♀ (soft) (7.1 mmCW) (ICMD002584, GenBank accession numbers MK415381,MK426941 and MK468904), 1 ovigerous ♀ (7.2 mm CW)(ICMD002585, GenBank accession numbers MK415383, MK426943and MK468906).

Description of male. Carapace (Figures 5A & 6A–C) circular (CL/CW = 0.99). Minimum and maximum width: 3.64 and 4.4 mm,respectively. Frontal margin projected anteriorly with densebrush of short setae medially. Dorsoanterolaterally, on bothsides of carapace, characteristic dense group of short and thicksetae (more than 30) present, superficially resembling tubercles(Figure 6B). Eyes clearly visible in dorsal view. Antenna short,with large basal segment followed by 4 segments, and with 2 or3 terminal setae on distal segment.

Third maxilliped (Figure 5B) with ischium and merus indistin-guishably fused, inner, outer margins with distal obtuse angle;palp 3-segmented with tuft of long simple setae from tip; propo-dus longer than carpus, dactylus inserted underneath propodus,on proximal half of ventral margin of propodus, apex not

reaching end of propodus. First thoracic sternite with anteriorborder concave.

Chelipeds (Figures 5C & 6A) similar. Palm (pal) slightlylonger than dactylus (d) (pal d−1 = 1.05), relationship length/width of palm = 1.1. Dactylus with well-developed tooth basally,smaller tooth present anteriorly; cutting edge of fixed finger ofpropodus with large tooth (in anterior position to that of mobilefinger), with about 15 short thick setae between apex of tooth.Tips of fingers curved.

Walking legs (Figure 5F–I, pereiopods 2–5) slender. P3, P4longer than P2, P5. P3 slightly longer than P4. Relative lengthP3 > P4 > P2 > P5. Merus of P2–P5 with short setae on dorsaland ventral sides. P3 and P4 with long swimming setae, in carpus(on lateral outer side), propodus and dactylus (on dorsolateralouter, and ventral sides), and some on merus of P5 (dorsodistaland ventral parts), carpus and propodus (dorsal and ventralparts). Without long swimming setae on P2 (Figure 5F).Relative lengths of merus are: P3 > P4 > P2 > P5; relationshipslength/width of merus leg P2 to P5 are 3.1, 3.1, 3.6 and 2.6respectively. Proportion of length of merus/length of dactylus ofP2 to P5 are 2.7, 2.4, 2.4 and 2.0 respectively. Propodus relativelengths are: P3 > P4 > P2 > P5. Dactylus of P2–P5 almost straight,with long curved distal part (claw), with small spiniform setae onventral concave faces, longer in P5. Dactylus of P3 and P4 longerthan others, subequal in length, P3≥ P4 > P2 > P5 (Figure 5F–I).

Male pleopod curving regularly, with apex narrowing progres-sively from base to tip (Figure 5D), with abundant setae from baseto apex, especially on concave face.

Pleon (Figure 5E) triangular, gradually narrowing, with 6somites and telson all free with rectilinear distal margin.

Description of hard female. These small females have long swim-ming setae on their pereiopods, similar to those of males. The gen-eral morphology is also very similar to that of males; the carapaceis circular, with the same characteristic dorsoanterolateral tufts ofshort setae (Figures 7A & 8A). Also, the morphology of chelipedsis similar to that of males. Relative length of walking legs is: P3 >P4 > P2 > P5. The dactyli of P3 and P4 are the longest and both areof similar lengths. The length/width relationships of the propodus(P2–P5) are: 2.18, 2.42, 2.33 and 1.73. Propodus/dactylus lengthrelationships (P2–P5) are: 1.60, 1.45, 1.56 and 1.33. Pleon onlyslightly wider than that of adult males (Figure 8B).

Description of soft females. Carapace (Figures 7B & 8C, E) subtra-pezoidal, wider than long (CL/CW = 0.76 to 0.81). Minimum and

Fig. 2. Relationship between Pinnotheres bicristatus sp. nov.carapace width and wet weight for each demographic cat-egory, and percentage represented by each category (piechart) in October 2017 and February 2018 surveys in Gulfof Cadiz.




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maximum width of carapace: 5.08 and 8.2 mm, respectively.Dorsal surface smooth, without dorsoanterolateral tufts of shortsetae. Front almost rectilinear, not projected, without setae(slightly convex in smallest soft females, AR-156: 5.08 mmCW). Small eyes, hardly visible in dorsal view. Antenna short,with large basal segment followed by 4 segments, and with 2 or3 terminal setae on distal segment.

Third maxilliped (Figure 7D) obliquely positioned on buccalcavity, not different from P. pisum (see Figure 5C in Becker &Türkay, 2010), ischium and merus indistinguishably fused,inner, outer margins with distal obtuse angle; palp 3-segmentedwith tuft of long simple setae at tip; propodus longer than carpus;dactylus inserted underneath propodus, almost subterminal, at orabout half length of ventral margin of propodus, apex reaches toor does not reach end of propodus.

Chelipeds (Figure 7E) similar. Palm longer than dactylus (pald−1 = 1.2–1.3), relationship length/width of palm = 1.2–1.3 (nar-rower than that of males and hard females). Dactylus with well-developed tooth basally, another on cutting edge of fixed fingerof propodus.

Walking legs (Figures 7F–I) slender. P3 longer than rest. Totalrelative length is: P3 > P4 > P2 > P5. Merus relative length: P3 >P4≈ P2 > P5. Length/width relationships of merus (P2–P5) are4.8, 5.1, 4.1 and 3.3 (P3 > P2 > P4 > P5), respectively. Propodus

relative lengths are: P3 > P4 > P2 > P5. Dactylus of P2–P5 almoststraight with curved distal part, with continuous row of tinyspines on ventral face of P2 and P4 (not in P3 and P5).Dactylus of P3 longer than rest (P3 > P4 > P2 > P5) (Figures 7B,G & 8C–E); more clearly in large specimens (Figures 7B, G &8C, D). Dactylus of P5 narrower than on others. Relationshipsbetween merus (m), propodus (p) and dactylus (d) lengthsof each P2 to P5 are as follow: m d−1 = 2.5, 2.0, 2.3 and1.6; p d−1 = 1.5, 1.4, 1.7 and 1.5.

Pleon, with 6 somites, very broad (Figures 7C & 8D); telsonwell separated with rectilinear distal margin, covering almostcompletely ventral side, even covering almost completely thethird maxillipeds (in large females), with setose margin andsmooth outer surface.

Etymology: The specific name bicristatus is an adjective (gen-der masculine as Pinnotheres) referring to the two ‘crests’ ordense tufts of setae located dorsoanterolaterally on the carapaceof males and hard females.

Habitat, host: All specimens collected were found insideAnomia ephippium collected between 11 and 47 m depth, on det-ritic bottoms.

Colouration in life: Males and hard females carapace withyellow-reddish colour with dark spots and with a characteristicpattern; it resembles a palm tree (Figures 6A & 8A), with a well-defined central ‘trunk’ and two pairs of lateral projections as‘leaves’, the posterior ones separated from this ‘trunk’. Behind,there are two well-defined rounded areas. The whole outline ofthis figure is reddish. Soft females with a pale-yellow colour:when females present developed gonads, they are reddish.

Discussion

MorphologyTaxonomically, within the genus Pinnotheres the species richnessis high, with a total of 52 species (WoRMS 2018) or 61 species(Palacios Theil et al., 2016), but only two species are known inthe area studied: P. pisum and P. pectunculi. In the past thisgenus was the most specious of the Pinnotheridae, but after sev-eral studies, especially those of Manning (1993a, 1993b) andCampos (1989, 1993, 2002), new genera have been recognisedfor many former Pinnotheres species. In contrast, no new speciesof Pinnotheres s. str. have been described since Pinnotheres tai-chungae Sakai, 2000.

Morphological differences with other European pinnotherids. InEuropean waters pinnotherid species have been known since

Fig. 3. Relationship between host shell size and Pinnotheres bicristatus sp. nov. popu-lation from Gulf of Cadiz. (A) Cumulative number of Anomia ephippium in increasingsize order vs cumulative number of pea crabs retrieved from them (plot on left topcorner) in October 2017 (solid line) and February 2018 (dotted line) surveys; numberof pea crabs by host (bar plot on right top corner) and pea crab prevalence by hostsize-groups (main bar plot) in October 2017(each left bar) and February 2018 (eachright bar) surveys. (B) Host shell size vs carapace width of pea crab for each crabdemographic category. ** and *, significance level of correlation coefficient of P <0.01 and P < 0.05, respectively.

Fig. 4. Topology of neighbour-joining tree based on concatenated 16S and Cox1mtDNA and H3 nuclear gene sequences, showing inferred phylogenetic relationshipswithin European Pinnotheridae, with American Zaops ostreus and Orthotheres barba-tus as outgroups. Numbers close to nodes indicate bootstrap support (only valuesabove 80% shown).




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Greek and Roman times (see Becker, 2010), although no descrip-tions of species were made until the 1700s. According to Ng et al.(2008) there are several old studies that were overlooked in thefirst description of Pinnotheres pisum (as Cancer pisum) byLinnaeus (1767) and subsequent studies. Scopoli (1763) describedCancer nutrix as a crab living inside Ostrea edulis from theAdriatic Sea. As Ng et al. (2008) pointed out, de Wulfen (1791)also described, from the Adriatic coasts, Cancer minutus, a speciesvery similar to Cancer nutrix and living inside O. edulis, but alsoin other bivalves. According to the very brief descriptions givenby these authors and considering that there are no type specimensextant available for checking, we believe that both taxa belong toP. pisum, mainly because this species is present in that part of theMediterranean Sea and is known to inhabit several different spe-cies of hosts, including O. edulis. At present, the complete distri-bution of Pinnotheres bicristatus sp. nov. is unknown and itcannot be excluded that this new species could be one of thosedescribed by Scopoli (1763) and de Wulfen (1791). We have,however, found only P. bicristatus sp. nov. inside Anomia ephip-pium, and not in any other bivalve species.

Assuming that C. nutrix can be assigned to P. pisum, and thatScopoli’s description in 1763 has priority with respect to that ofLinnaeus (1767), the correct name for P. pisum would bePinnotheres nutrix (Scopoli, 1763). Prior to being more conclu-sive, as suggested by Ng et al. (2008), it could be necessary toassign neotypes to these species by collecting fresh specimenshosted by O. edulis from the Adriatic Sea and also take intoaccount that Ŝtevčić (1990) also mentioned N. pinnotheres insideOstrea edulis in this region. This is, however, beyond the scope ofthe present study.

Two other species have been described from European waters,Pinnotheres ascidicola Hesse, 1872 and P. marioni Gourret, 1888.Recently, Becker & Türkay (2010) made a detailed study ofEuropean pinnotherids and concluded that both species are

junior synonyms of N. pinnotheres, based on the original descrip-tions and figures (because there is no type material extant) andtogether with the fact that the hosts of both species are ascidians.Based on the same evidence and the previous paper by Gourret(1887), we have also concluded that they are junior synonymsof N. pinnotheres, and we are confident neither can be P. bicrista-tus sp. nov.

When comparing P. bicristatus sp. nov. with the recognizedspecies of European pinnotherids, males and hard females fromall those species can be easily differentiated by two peculiar char-acteristics on the cephalothorax: (a) a pair of dorsoanterolateraltufts of curved setae (short and very close to each other) thatresemble two tubercles (Figures 5A, 6B & 7A), not described inthe previous known European species or in other species forwhich we have evidence; and (b) the orange-reddish ‘palm tree’marking covering the dorsal surface (Figures 5A, 6A, C, 7A &8A). Concerning this latter feature, males of P. pisum show asomewhat similar, although different, marking in which the‘trunk of the palm tree’ is missing and the colour is yellow-orangish (see pictures in the Crustikon – Crustacean photo-graphic website – Tromsø Museum, University of Tromsø,http://archive.li/rHrj6, by Cédric d’Udekem d’Acoz; andFigure 1B in Becker & Türkay, 2010).

Pinnotheres bicristatus sp. nov. can be differentiated from theAfrican species Afropinnotheres monodi by the morphology ofthe third maxilliped: in A. monodi the propodus is shorter thanthe carpus (this is not the case in the genus Pinnotheres andNepinnotheres), and the dactylus extends well beyond the end ofthe propodus (see A. monodi Figure 1A, in Manning, 1993a),while these features are not present in the rest of European species(at most, the dactylus extends only very slightly in N. pinnotheres;see Figure 3C in Becker & Türkay, 2010). Also, in A. monodi, thedorsal surface of the carapace in males and hard females is finelypubescent.

Soft females of the new species and those of Nepinnotherespinnotheres can be differentiated easily by the apex of the dactylusof the third maxilliped, which reaches or exceeds the distal part ofthe propodus in N. pinnotheres (see Figure 24B in Manning,

Fig. 5. Pinnotheres bicristatus sp. nov. Holotype male IEO-CD-ICMAN/2419 (4.4 mmcarapace width). (A) Carapace, dorsal view and figure pattern; (B) third maxilliped,outer and inner views; (C) right cheliped, outer view; (D) male first gonopod; (E)pleon, outer view; (F–I) outer views of the second to fifth right pereiopods. Scalebars: (A, C), (E–I), 1.0 mm; (B, D), 0.5 mm.

Fig. 6. Pinnotheres bicristatus sp. nov. Holotype male IEO-CD-ICMAN/2419 (A, C) dor-sal view, showing colour pattern; (B) detail of dorsoanterolateral carapace regionwith tufts of short curved setae.



http://archive.li/rHrj6

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1993a; Figure 3C in Becker & Türkay, 2010), but not in P. bicris-tatus sp. nov. (Figure 7D). The carapace morphology is subglob-ular, with width greater than length, in N. pinnotheres, whereas itis subtrapezoidal in P. bicristatus. However, both species possess abroad triangular tooth at about mid-length of the fixed finger ofthe chelipeds (which does not exist, or else is very small, in P.pisum), and the dactylus lengths of P5 are more than half thelength of their propodus. However, in the new species, the dacty-lus of P3 is clearly longer than those of the rest of its walking legs,a difference especially pronounced in large females (Figures 7B, G& 8C, D).

Soft females of Pinnotheres pectunculi have a subtrapezoidalcarapace with an approximately rectilinear front that is not pro-jected (see Figure 25 in d’Udekem d’Acoz, 1989 and Figure 9A,B in the present work) as in P. bicristatus sp. nov. (Figures 7B& 8C, E) (in N. pinnotheres the frontal area is a little projected,bilobed by a median incision, whereas in P. pisum it is slightlyrounded). They can also be easily distinguished by the dactyliof the walking legs, which are very different. Thus, in P. pectunculithey are very short (d’Udekem d’Acoz, 1989) (Figure 9A, B) (evenshorter than those of P. pisum), while they are long in the newspecies, especially in large soft females, whose legs are longer,and thinner (stylized) than those of hard individuals.

With regards to morphology, Pinnotheres pisum is the speciesclosest to P. bicristatus sp. nov. They can be separated by the rela-tive length of the dactyli of the walking legs; these are shorter(clearly less than half as long as the propodus) and more curvedin P. pisum (see Figure 4A in Becker & Türkay, 2010 and Figures7B, F–I & 8C–E in present study). In addition, in soft females ofP. pisum, the dactyli are of almost equal length on all walking legs(see Figure 5 in Becker & Türkay, 2010), while in the new speciesthe dactyli is clearly longer on the third pereiopod (Figure 7G). Inaddition, the carapace in P. pisum is subglobular (see Figure 5 in

Becker & Türkay, 2010), not subtrapezoidal as in the new species(Figures 7B & 8C, E).

Identification key for European Pinnotheridae.

1. Mxp3 with propodus conical, tapering distally, shorterthan carpus. Dactylus inserted at base of ventral margin ofpropodus, overreaching end of propodus . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . Afropinnotheres monodi

Mxp3 with propodus oval or spatulate, longer than carpus.Dactylus inserted at mid-length or at base of ventral margin ofpropodus but apex not extending to end of propodus . . . . . . 2

2. Carapace of hard specimens (males and females) with dor-soanterolateral tuft of setae. Soft females with subtrapezoidalcarapace, dactylus of P3 longer than others, dactylus of P5 nar-rower, with a relation propodus/dactylus length of1.5 . . . . . . . . . . . . . . . . . . Pinnotheres bicristatus sp. nov.

Carapace of hard specimens (males and females) without dor-soanterolateral tuft of setae. Soft females with subtrapezoidal orsubglobular-rounded carapace, dactylus of P2-P5 more or lesssimilar in length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Carapace and pereiopods covering with tomentum. P2–P5with long dactylus, that of P5 slightly longer than others,approximately as long as three-quarters of propodus.Dactylus of Mxp3 styliform or spatulate, inserted at or nearmidlength of ventral margin of propodus, apex extending toor overreaching end of propodus . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .Nepinnotheres pinnotheres

Carapace and pereiopods smooth. P2–P5 with short dactylus.Dactylus of Mxp3 styliform, inserted at base of ventral margin ofpropodus, apex not extending to end of propodus . . . . . . . . 4

Fig. 7. Pinnotheres bicristatus sp. nov. (A) Paratype hard female (3.27 mm carapacewidth) IEO-CD-ICMAN/2420, dorsal view; (B–I) Paratype ovigerous female (8.2 mmcarapace width), IEO-CD-ICMAN/2440, from Barbate (setae not represented): (B) cara-pace in dorsal view; (C) ventral view; (D) third right maxilliped (outer view); (E) chelaof first left pereiopod (outer view); (F–I) outer view of second to fifth left pereiopod.Scale bars: (A–C), (E–I), 1.0 mm; (D), 0.5 mm.

Fig. 8. Pinnotheres bicristatus sp. nov. (A, B) Paratype hard female IEO-CD-ICMAN/2420, habitus: (A) dorsal view; (B) ventral view. (C, D) Paratype ovigerous femaleIEO-CD-ICMAN/2440, habitus, dorsal and ventral views (left and right respectively);(E) Paratype soft female (7.88 mm carapace width), IEO-CD-ICMAN/2428, from Gulfof Cadiz, in dorsal view (right) and detail of walking left legs in ventral view (left).




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4. Carapace of soft females subglobular-rounded. Eyes visible ondorsal view. Dactylus of walking legs (P2–P5) with short,curved tips, less than half as long as propodus. Fixed fingerof chela (propodus of P1) with single tooth . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pinnotheres pisum

Carapace of soft females subtrapezoidal. Small eyes. Walkinglegs (P2–P5) slender with short, pointed, curved dactylus, consid-erably less than half as long as propodus. Fixed finger of chela(propodus of P1) with a well-developed basal tooth followed byadditional small ones . . . . . . . . . . . . . . Pinnotheres pectunculi

All males of the European pinnotherid species can be distin-guished by the morphology of the first gonopod (seeFigure 9C–G). Each first gonopod has a different degree ofcurvature, from that of A. monodi, which is practically straightand only lightly curved at the apex (Figure 9C) to that of N.pinnotheres, which is almost L-shaped (Figure 9E); inPinnotheres spp., it has an intermediate shape. The first gono-pod of P. pisum is straight and wide over almost its total length,but its distal part is narrowed and curved (Figure 9D), while inP. pectunculi and P. bicristatus sp. nov. the gonopod is curvedalong its full length; however, the curvature of gonopod is morepronounced in P. pectunculi (Figures 6C, D in Becker &

Türkay, 2010, Figure 9F in present study) than in P. bicristatus(Figure 9G).

The first gonopod of A. monodi was not described when thespecies was described by Manning (1993a), because the onlymale specimen lacked these appendages; therefore, this is thefirst time that the gonopod of A. monodi has been illustrated(Figure 9C).

About the specific host and associated pinnotherid species. Untilnow, only four species of pinnotherid crabs have been reportedin association with bivalves of the genus Anomia (see Schmittet al., 1973). Pinnotheres gordoni Shen, 1932, with distributionin China and Japan (Silas & Alagarswami, 1967), has beenreported as living inside Anomia cytaeum Gray, 1850 (syn.Anomia lischkei Dautzenberg & Fischer, 1907), which has a gen-eral distribution in the waters of China, Japan and India (Higoet al., 1999; Souji & Radhakrishna, 2015). According to thedescription by Shen (1932), P. gordoni has pubescent chelipedsand walking legs, P4 being the longest pereiopod, while the dac-tylus of P5 is subequal to the propodus of the same leg. In thesecharacters, it is clearly different from P. bicristatus.

Two pinnotherid crabs from West Atlantic waters, namelyTumidotheres maculatus (Say, 1818), known to occur fromMassachusetts to Argentina (Kruczynski, 1974), and Zaops ostreus(Say, 1817), present from Massachusetts to Cuba, Guadalupe andBrazil (Palacios et al., 2016), have been found inside Anomia simplexd’Orbigny 1853. This bivalve has a general distribution rangingfrom Nova Scotia to Mexico and the Caribbean (Rosenberg,2009). Given that T. maculatus and Z. ostreus belong to differentgenera from that of Pinnotheres bicristatus sp. nov., the differencesare major (see Campos, 1989; Manning, 1993a, 1993b). Amongthese differences to be noted are the size of the dactylus of P5,which is longer than the dactyli of P2–P4 in T. maculatus and Z.ostreus; in P. bicristatus sp. nov., P3 has the longest dactylus.

Finally, the fourth species reported as being hosted by Anomia isthe European Pinnotheres pisum, which has a general distributionfrom Norway to the Mediterranean Sea and Canary Islands(Triay-Portella et al., 2018), and has been recorded twice inAnomia ephippium (Miranda y Rivera, 1921; Delongueville &Scaillet, 2002). This bivalve is distributed from Norway toMorocco, and Angola, including also the Mediterranean Sea(MolluscaBase 2018, accessed through WoRMS). The oldest recordin A. ephippium is one by Miranda y Rivera (1921), from Malaga(Alboran Sea) at 30 m depth, which mentioned the presence oftwo females of P. pisum hosted in A. ephippium, and a second(and the most recent known to the authors) is one female reportedin Rota (Gulf of Cádiz) by Delongueville & Scaillet (2002). Bothlocations are within the area sampled in the present study.Unfortunately, we cannot confirm these identifications because thetwo specimens from Malaga (Miranda y Rivera, 1921) no longerexist in the collection where they were deposited (IEO), and thephotograph of the female from Rota in Delongueville & Scaillet(2002) does not allow a correct identification. However, we have ana-lysed a large amount of material of A. ephippium from the Gulf ofCádiz and Málaga and the only pea crab found living in associationwith this bivalve is Pinnotheres bicristatus sp. nov. On this evidence,it is very likely that the records of P. pisum in A. ephippium byMiranda y Rivera (1921) and Delongueville & Scaillet (2002) areactually P. bicristatus sp. nov. The present new crab species probablyhas a wider distribution throughout European and African waters(North-east Atlantic and Mediterranean Sea), occurring whereverA. ephippium is found.

With these two possible exceptions of Miranda y Rivera (1921)and Delongueville & Scaillet (2002), Pinnotheres bicristatus sp.nov. had not been previously recorded in European waters. Thisoversight of its presence could be explained if P. bicristatus sp.

Fig. 9. Pinnotheres pectunculi: (A) soft female of San Roque, Cádiz, habitus in dorsalview (left) and details of left walking legs (right); (B) soft female from Marbella,Málaga, habitus in dorsal view and details of propodus and dactylus of right walkinglegs P3–P5. Schematic drawings of first gonopod of European pinnotherids (setaeremoved): (C) Afropinnotheres monodi (IEO-CD-ICMAN/2442, Gulf of Cadiz); (D)Pinnotheres pisum (from Barbate Cadiz, June1994, Manjón-Cabeza & García Raso,1988); (E) Nepinnotheres pinnotheres (from Malaga, 1978 within Atrina pectinata); (F)Pinnotheres pectunculi (figure modified from Becker & Türkay, 2010); (G) Pinnotheresbicristatus sp. nov. (holotype IEO-CD-ICMAN/2419). Scale bars: (C–E), 1.0 mm; F (fromBecker & Türkay, 2010), 200 µm; G, 0.5 mm.




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nov. is not native to the region of study and its introduction intoEuropean A. ephippium had occurred recently. However, the rela-tive high abundance of P. bicristatus in A. ephippium from theGulf of Cadiz and the distribution range presently known supportinstead the hypothesis that this new pinnotherid is a native spe-cies, whose presence has been omitted because its host A. ephip-pium, which is not a commercial species, has been rarely collectedand, consequently, has not been well studied. Besides, morpho-logical and molecular results of this study suggest that P. bicrista-tus sp. nov. is a native species since it is closely related to P. pisumand P. pectunculi: it has been shown that the geographic distribu-tion of species plays an important role in their phylogenetic rela-tionships (Cuesta et al., 2012), and it would be more difficult tofind this molecular proximity between this new pinnotherid andthe other two native species if the former was an introducedexotic species. For this reason, molecular data would support anAtlantic-Mediterranean origin of P. bicristatus sp. nov.

Pinnotheres bicristatus sp. nov. population hosted by AnomiaephippiumThe high prevalence of Pinnotheres bicristatus sp. nov. in thecommon saddle oyster Anomia ephippium populations of theGulf of Cádiz suggests that this crab forms a well-establishedpopulation in the area. Although the smallest sex-indeterminateindividuals of P. bicristatus were not found inside A. ephippium,the presence of at least 20% of males, together with hard andreproductive females, in this host makes it possible that a stablepopulation of this crab exists even without any additional host.For other pinnotherid species, sex ratios biased towards a lowerproportion of males have been explained by possible predationwhen males leave their hosts to find mates (Trottier & Jeff,2012). However, it is also plausible that other bivalve specieswere facultative hosts of males and of smaller specimens thatwere not found inside A. ephippium, as is the case forAfropinnotheres monodi found in the same area (Drake et al.,2014; Pérez-Miguel et al., 2019). The mussel Mytilus gallo-provincialis is the primary host of the soft (reproductive) femalesof A. monodi, whereas other bivalves of small size (such asCerastoderma spp. and Scrobicularia plana) are the primaryhosts of hard females and males. Notwithstanding, about 15.8%of A. monodi crabs hosted by mussels were males (Drake et al.,2014; Pérez-Miguel et al., 2019). If males and hard females ofP. bicristatus were using another bivalve species as primaryhost, it would probably be the case that this unknown host speciesis also subtidal, since we did not collect any specimens of this crab(Pérez-Miguel et al., 2018) during an exhaustive sampling ofintertidal bivalves along the coasts of the Iberian Peninsula in2016 and 2017.

Since the timing and duration of reproductive periods is influ-enced by water temperature and/or the light-dark cycle (Sastry,1983), it is reasonable to assume that Pinnotheres bicristatus sp.nov. has one single reproductive period throughout the knowndistribution area (Figure 1). Currently, the available informationon this topic is the presence of larval stages (zoeae and megalo-pae) found in November 2013 (Marco-Herrero et al., 2018), oneovigerous female found in July 1995 in the Gulf of Cádiz, andone ovigerous female found in April 2017 in the Alboran Sea(this study). According to this information, P. bicristatus sp.nov. would have a long reproductive period extending from springto autumn. Furthermore, there was a clear predominance ofreproductive females with well-developed gonads both inOctober 2017 and in February 2018, whereas ovigerous femaleswere absent, suggesting that a slowdown of the final gonadaldevelopment occurred during the cold period and that spawningdoes not take place until the water temperature starts to rise.Concerning crab pairs, there was a remarkable increase between

October 2017 (3.3% of hosts infested) and February 2018(26.7% infested). A positive relationship between crab prevalenceand the proportion of multiple infested hosts has been found inother pinnotherid crabs (Asama & Yamaoka, 2009; Mena et al.,2014; Pérez-Miguel et al., 2019). Thus, it is not possible to rejectthe view that the increased multiple infestations may be partiallyassociated with the increase in the proportion of hosts infested byP. bicristatus sp. nov. observed between October 2017 andFebruary 2018: on average, 55.6 vs 77.7% of host specimensinfested, respectively. However, since heterosexual pairs were col-lected more frequently than expected by chance, a more probableexplanation of the increase of male-female pairs inside a singlehost in February was the increase of encounters for mating justbefore starting the spring spawning period. Mating that takesplace inside the hosts, where soft female crabs remain stationary,and with males visiting various bivalves hosting soft females, hasbeen suggested for other pinnotherid species (Soong, 1997;Ocampo et al., 2012).

Females of Pinnotheres bicristatus reached larger sizes thanmales, a sexual dimorphism that is very common among peacrabs (e.g. see Soong, 1997; Ocampo et al., 2012; Drake et al.,2014). Concerning the relationship between host size and crabsize, a strong correlation was found between Anomia ephippiumsize and ovigerous female size of P. bicristatus sp. nov.; this sug-gests that availability of space inside a host is a relevant factor indetermining the final size of the crab in its sedentary phase (i.e.soft females). Although a significant correlation between hostsize and male crab size was also found in the population studied,the correlation was weaker than for females. Furthermore, foreach specific host size, male crabs were smaller than femaleshosted by it (Figure 3B). A similar sex-dependent pattern in therelationship between host size and the symbiotic/parasitic crabsize has been found previously for other pinnotherid species(Soong, 1997; Kane & Farley, 2006; Ocampo et al., 2012;Drake et al., 2014). As stated by Soong (1997), there is a closercorrelation in females than in males, between crab size andhost size; this may be due to the longer association betweena female crab and its host (females stay longer in the samehost than males do). Considering all the information available,we hypothesize that, initially, P. bicristatus sp. nov. hard femalesdo not make their selection of host by size (there is no correl-ation between the size of a hard female and that of its host).However, after their metamorphosis to soft females, they cannotleave the host and their growth might be limited by the host size,which explains the observed strong correlation in size betweensoft females of P. bicristatus sp. nov. and the specimens ofA. ephippium hosting them. In the case of the smaller andmore motile males, they might not be limited by host size but,as they may change hosts frequently, larger males may avoidrelatively small (growth-limiting) hosts and this behaviourcould explain the significant but low correlation in size foundbetween hosts and male crabs.

Final remarkAs previously commented, Pinnotheres bicristatus sp. nov. prob-ably has a wider distribution throughout European and Africanwaters linked to the occurrence of its host Anomia ephippium.However, due to the scarce studies on this bivalve species, theexistence of P. bicristatus sp. nov. could have been overlookeduntil now. Therefore, we would like to underline the importanceof studying the plankton-collected larvae in combination withDNA analyses, to discover, as in the present case, hidden crypticnew species or non-indigenous species introduced in new areas.

Author ORCIDs. Jose A. Cuesta, 0000-0001-9482-2336; J. E. García Raso,0000-0003-3092-9518



https://orcid.org/

http://orcid.org/0000-0001-9482-2336

http://orcid.org/0000-0003-3092-9518


https://doi.org/10.1017/S0025315419000018


Acknowledgements. We are grateful to all participants in the ARSA andMEDITS surveys run by the Spanish Institute of Oceanography (IEO), fromwhich many of the samples come, particularly to their chief scientists(Ignacio Sobrino and Cristina García). We want to thank Carlos Sánchez(ICMAN) for laboratory work. Thank are also due to Maria EugeniaManjón-Cabeza for sharing specimens collected in Barbate samples. Wewant to thank two anonymous referees, and especially to Peter Ng for hisdetailed corrections, comments and suggestions that clearly improved themanuscript, and Royston Snart for the linguistic revision of the English text.

Financial support. This study was partially funded by the Spanish ‘Ministeriode Economía y Competitividad (MINECO), Plan Nacional I + D’ and theEuropean FEDER funds through project AFROBIV (CGL2014-53557-P), andthe Project PB92-0415, supported by the Spanish M.E.C, DGICYT funds.

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